The Role And Underlying Molecular Mechanisms Of Nasal Commensal Viruses In The Regulation Of Allergic Rhinitis

Allergic rhinitis（AR）is a non-infectious inflammation of the nasal mucosa mediated by specific immunoglobulin E（Ig E）,with an incidence rate of 10%-20%and a rising trend annually.AR can occur at any age and its onset and development are related to the subject’s constitution,lifestyle habits,and environment.Typical symptoms of AR include sneezing,rhinorrhea,nasal itching,and nasal congestion,often accompanied by various complications such as asthma,sinusitis,and otitis media.Severe allergic rhinitis can affect the patient’s quality of life,sleep,and work performance.Currently,the main treatment methods for AR include symptomatic treatment,including medication and surgical treatment,which cannot cure the condition;and specific immunotherapy,which has a long duration（2-3 years）,is cumbersome to administer,has poor patient compliance,and uncertain efficacy.Therefore,discovering more effective and safer drugs for the treatment of AR is of significant importance.In recent years,studies have shown a significant correlation between the imbalance of symbiotic viruses and the severity of inflammatory diseases.The concept of"symbiotic viruses"was first proposed in the 19th century to describe lichens,which are symbiotic organisms consisting of fungi and algae.Broadly speaking,symbiosis refers to two different species living closely together,and narrowly,based on the relationship between virus and host,symbiotic relationships can be classified into three types:antagonistic,mutualistic,and commensal.In this study,we adopt the latter definition,where commensal viruses do not lead to severe disease states in the host organism.These viruses are termed"commensal viruses."However,they may alter the host’s physiological state to maintain host homeostasis or increase host susceptibility to disease,regardless of their infectious nature as viruses.The mammalian virome includes viruses infecting eukaryotic cells,bacteriophages and genetic elements derived from viruses integrated into the host chromosome.With the advancement of second-generation sequencing technologies,metagenomic sequencing enables the detection of various commensal viruses residing on and within the human body.Studies have shown that,on average,each healthy individual carries no fewer than 10 commensal viruses,and due to technological limitations,the actual number of virus species present may be much higher than those detected.Commensal viruses play a crucial role in regulating the immune homeostasis of the host organism.Research indicates that commensal human papillomavirus（HPV）within the body can suppress the occurrence of skin cancer by enhancing the host immune response.Similarly,commensal viruses in the gut contribute to maintaining the quantity of intraepithelial lymphocytes via noncanonical RIG-I-like receptor（RLR）signaling pathways,reducing susceptibility to inflammatory bowel diseases in hosts.However,studies have shown that commensal viruses may render individuals susceptible to specific diseases in certain genetic backgrounds.A study revealed that persistent presence of Murine norovirus（MNV）combined with low-level expression of autophagy-related 16-like protein 1（ATG16L1）can lead to susceptibility to Crohn’s disease in hosts.However,the presence of MNV alone or low-level expression of ATG16L1 does not predispose hosts to Crohn’s disease.This illustrates the dual role of commensal viruses in regulating host immunity-they can maintain host homeostasis while potentially rendering the host susceptible to certain diseases.These findings collectively underscore the crucial role of commensal viruses in modulating the host immune system.Particularly,the nasal cavity,as the primary gateway for the body’s interaction with the external environment,harbors a vast array of commensal microorganisms,yetthe role of nasal commensal microorganisms in regulating the host immune system remains largely unexplored.Studies have shown the presence of abundant viral communities in the respiratory tracts of healthy individuals.Therefore,in this study,we focus on nasal cavity commensal viruses to investigate their mechanistic roles in the occurrence and development of allergic rhinitis,aiming to identify novel targets for intervention and treatment of allergic rhinitis.We found that nasal commensal viruses can promote the occurrence and development of allergic rhinitis.When inducing allergic rhinitis in mice using ovalbumin（OVA）,the presence of commensal viruses induced a subset of neutrophils with high expression of interferon-stimulated genes（ISGs）,termed ISG^highneutrophils.These ISG^high neutrophils promote the occurrence and development of allergic rhinitis by generating neutrophil extracellular traps（NETs）.PartⅠ.The commensal viruses in the nasal cavity promote the onset and progression of allergic rhinitis.To investigate the regulation and mechanism of nasal commensal viruses on the occurrence and development of allergic rhinitis,this study first used metagenomic sequencing to determine the presence of commensal viruses in the nasal cavities of C57BL/6mice housed in Specific-Pathogen Free（SPF）conditions.We employed ribavirin to clear the nasal commensal viruses in mice,followed by OVA induction to induce allergic rhinitis.We found that clearing nasal commensal viruses significantly improved the severity of allergic rhinitis in mice.To explore the mechanism by which commensal viruses exacerbate allergic rhinitis,we used flow cytometry to assess immune cell infiltration in the nasal mucosa of mice.Mice were divided into two groups:one group had nasal commensal viruses cleared and were induced with allergic rhinitis using OVA on ribavirin treatment（OVA+Ribavirin group）,while the other group had nasal commensal viruses intact and were induced with allergic rhinitis using OVA without ribavirin treatment（OVA+Vehicle group）.Results showed that compared to the OVA+Ribavirin group,mice in the OVA+Vehicle group exhibited a significant increase in neutrophil levels in the nasal mucosa,suggesting that neutrophils may play an important role in the exacerbation of allergic rhinitis by nasal commensal viruses.To further explore the specific mechanism by which nasal commensal viruses exacerbate allergic rhinitis,we obtained nasal mucosa from four groups of mice:Vehicle group,Ribavirin group,OVA+Vehicle group,and OVA+Ribavirin group.CD45⁺cells were isolated from the nasal mucosa and subjected to single-cell sequencing.The results of single-cell sequencing,consistent with the flow cytometry results,showed that compared to mice in the OVA+Ribavirin group,mice in the OVA+Vehicle group exhibited a significant increase in neutrophil levels in the nasal mucosa.PartⅡ.Commensal virus induces an elevated level of ISG^high neutrophils.We conducted an analysis of the single-cell sequencing data,revealing that neutrophils could be divided into 7 subsets based on gene expression profiles,namely N01-Cxcl3^highEro1l^high,N02-Siglecf^highTgm2^high,N03-Cxcl3^highRetnlg^high,N04-Siglecf^highMrpl52^high,N05-Cxcl3^highIsg15^high,N06-Siglecf^highGpc3^high,and N07-Ngp^high.Furthermore,based on highly expressed genes,they were classified into 3 major subsets:Cxcl3^high（N01,N03,N05）neutrophil subset,Siglecf^high（N02,N04,N06）neutrophil subset,and Ngp^high（N07）neutrophil subset.According to the single-cell sequencing data,we found a significant increase in the Cxcl3^high subset of neutrophils in the OVA+Vehicle group.We observed that compared to the Siglecf^high subset,the Cxcl3^high subset of neutrophils exhibited significantly increased degranulation,myeloid cell chemotaxis ability,and interferon signaling pathway scores,while neutrophil cytotoxicity was significantly decreased,indicating a response to interferon in the Cxcl3^high subset.Furthermore,compared to the Siglecf^high subset,the Cxcl3^high subset showed significantly increased expression of the interferon-stimulated gene Ifit1.Consequently,we labeled the Cxcl3^high subset with IFIT1flow cytometry antibody and named this population as ISG^highneutrophils.Enrichment analysis of genes highly expressed in the ISG^high subset revealed their involvement in processes such as reactive oxygen species regulation,metabolism,oxidative stress response（ROS）,extracellular signal-regulated kinase 1（ERK1）and extracellular signal-regulated kinase 2（ERK2）cascade reactions,endoplasmic reticulum stress,type I interferon response,etc.These processes have been previously associated with the formation and release of neutrophil extracellular traps（NETs）,indicating that ISG^high neutrophils may be more prone to generate and release NETs.To explore whether commensal virus-induced neutrophil subsets have different origins from other subsets,we performed pseudo-temporal analysis of neutrophil differentiation and development using single-cell sequencing data.Neutrophils were classified into five developmental states,with two distinct differentiation trajectories observed.Analyzing the expression changes of differential genes along the pseudo-temporal trajectory,we found that the Siglecf^high（N02,N04,N06）subset resided in the late stage of neutrophil differentiation and development,while the Cxcl3^high（N01,N03,N05）,namely ISG^high subset,occupied the early to middle stages of cellular differentiation and development,indicating their more immature status.This suggests that in the presence of commensal viruses,there are more immature ISG^high neutrophils that migrate to inflammatory sites.The body primarily recognizes virus-associated molecular patterns through pattern recognition receptors,including RIG-I-like receptors,Toll-like receptors（TLRs）,cyclic GMP-AMP synthase（c GAS）,which activate downstream signaling pathways,leading to the synthesis and release of inflammatory cytokines and type I interferons.To explore which pattern recognition receptor recognizes nasal commensal viruses,exacerbating allergic rhinitis,we used Rigi^-/-,Tlr3^-/-,Cgas^-/-,Sting1^-/-mice for OVA sensitization,with C57BL/6wild-type mice as controls.The results showed that pattern recognition receptor knockout mice exhibited similar phenotypes to Ribavirin-treated mice,with significantly alleviated symptoms of allergic rhinitis.Flow cytometry analysis of neutrophils and ISG^highneutrophil levels in mouse nasal mucosa showed a similar phenotype to the Ribavirin-treated group,with significantly reduced levels in pattern recognition receptor knockout mice.These results suggest that nasal commensal viruses exacerbate allergic rhinitis through the RIG-I/TLR/c GAS-STING pathway,while also inducing elevated levels of ISG^high neutrophils.Since activation of the RIG-I,TLR3,and c GAS-STING signaling pathways can all initiate the transcription and production of type I interferons,it is likely that type I interferons play a crucial role in the exacerbation of allergic rhinitis by nasal commensal viruses.Therefore,we utilized interferon regulatory factor 3（IRF3）knockout mice and interferon alpha/beta receptor 1（IFNAR1）knockout mice,subjected to OVA-induced allergic rhinitis,with C57BL/6 wild-type mice as controls.The results demonstrated that compared to the control group,both Irf3^-/-and Ifnar1^-/-mice showed significantly alleviated allergic rhinitis phenotypes,consistent with the Ribavirin-treated group mice.Furthermore,we replenished type I interferons on the basis of OVA-induced allergic rhinitis.C57BL/6 wild-type mice were divided into two groups:one group was replenished with IFNα1b based on OVA-induced allergic rhinitis（IFNα1b group）,while the other group was replenished with an equal volume of phosphate-buffered saline devoid of IFNα1b（Vehicle group）based on OVA-induced allergic rhinitis.The results showed that compared to the Vehicle group mice,the IFNα1b group mice exhibited more severe symptoms of allergic rhinitis,with significant increases in inflammatory cytokines IL-1β,IL-4,IL-5,IL-13,IL-6,IL-17 at the m RNA level,and interferon-stimulated gene CXCL10.Flow cytometry analysis of neutrophil levels in mouse nasal mucosa revealed a significant increase in ISG^highneutrophils in the IFNα1b group mice.These findings suggest that nasal commensal viruses may exacerbate allergic rhinitis through the RIG-I/TLR3/c GAS-STING-IRF3-Type I IFN-IFNAR1 signaling pathway,while also inducing elevated levels of ISG^high neutrophils.PartⅢ.ISG^high neutrophils promote the onset and progression of allergic rhinitis.Gene enrichment analysis revealed that transcripts enriched in ISG^high neutrophils are mainly associated with the regulation of ROS,metabolic processes,oxidative stress responses,ERK1 and ERK2 cascade reactions,endoplasmic reticulum stress,and type I interferon responses.Previous studies have suggested that these processes are related to the formation and release NETs.Therefore,ISG^high neutrophils may be more inclined to generate and release NETs.Flow cytometry analysis of ROS production in neutrophils showed that nasal commensal viruses induce neutrophils to produce more ROS.Immunofluorescence detection of neutrophil extracellular traps showed a significant increase in NETs formation induced by nasal commensal viruses.To investigate whether NETs are produced by interferon-induced ISG^high neutrophils,we replenished IFNα1b while sensitizing mice with OVA to induce neutrophils to become ISG^high neutrophils.NETs formation was then assessed.The results showed a significant increase in NETs production in the nasal mucosa neutrophils of mice with IFNα1b replenishment,suggesting a propensity of ISG^highneutrophils to produce NETs.To validate this hypothesis,we utilized Ifnar1^-/-mice,induced with OVA to develop allergic rhinitis,with wild-type C57BL/6 mice as controls.The results demonstrated a significant reduction in NETs production upon knockout of the type I interferon receptor.These findings indicate a predisposition of ISG^high neutrophils to produce NETs.To explore whether NETs are correlated with the occurrence and development of allergic rhinitis,we used the NETs generation inhibitor neutrophil elastase inhibitor（NEi）to suppress NETs formation,as well as employed deoxyribonuclease I（DNase I）to degrade NETs.The results showed that clearing NETs significantly alleviated allergic rhinitis symptoms,manifested as reduced symptoms of allergic rhinitis and decreased infiltration of eosinophils in the nasal mucosa,suggesting that NETs promote the occurrence and development of allergic rhinitis,and clearing NETs significantly alleviated allergic rhinitis symptoms.In summary,this study discovered that nasal commensal viruses induce an increase in ISG^highneutrophil levels through the RIG-I/TLR3/c GAS-STING-IRF3-Type I IFN-IFNAR1pathway.ISG^high neutrophils,in turn,promote the occurrence and development of allergic rhinitis by producing NETs.By linking innate immunity with allergic diseases through commensal viruses,this study enhances our understanding of the regulatory network of innate immunity and opens up a new research avenue by considering nasal commensal viruses in the treatment of allergic rhinitis.Furthermore,commensal viruses present a potential novel target for the diagnosis and treatment of allergic rhinitis.